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We present a fast mapping-based algorithm to compute the mappability of each region of a reference genome up to a specified number of mismatches. Knowing the mappability of a genome is crucial for the interpretation of massively parallel sequencing experiments. We investigate the properties of the mappability of eukaryotic DNA/RNA both as a whole and at the level of the gene family, providing for various organisms tracks which allow the mappability information to be visually explored. In addition, we show that mappability varies greatly between species and gene classes. Finally, we suggest several practical applications where mappability can be used to refine the analysis of high-throughput sequencing data (SNP calling, gene expression quantification and paired-end experiments). This work highlights mappability as an important concept which deserves to be taken into full account, in particular when massively parallel sequencing technologies are employed. The GEM mappability program belongs to the GEM (GEnome Multitool) suite of programs, which can be freely downloaded for any use from its website (http://gemlibrary.sourceforge.net).

A species-specific composite pain scale is a prerequisite for adequate pain assessment. The aim of this study was to develop a multidimensional pain scale specific to rabbits ( Oryctolagus cuniculus ) called the Bristol Rabbit Pain Scale (BRPS). The scale was developed over five phases using a unique combination of methods: focus groups and behavioural observation. The first two phases aimed at identifying descriptors to describe a rabbit in pain, and then reducing their number, both using focus groups. A total of 72 pain descriptors were grouped under six categories (Demeanour, Posture, Facial expression, Attention to the painful area, Audible and Other) and ‘No pain’ descriptors were added. The third phase aimed to confirm, through video observation of rabbits, the categories and descriptors previously described, to reject those terms that were ambiguous, and identify any new descriptors that had not been included in the previous list of descriptors. This led to the rejection of the categories Audible and Attention to the painful area and of 34 descriptors. Seven new descriptors were identified. The last two phases constructed the final format of the BRPS by refining the categories, ranking the descriptors on an ordinal scale and testing the internal reliability of the scale using Cronbach’s alpha test. This led to a composite pain scale of six categories (Demeanour, Posture, Locomotion, Ears, Eyes and Grooming) with four intensities of pain (0, 1, 2, and 3), a total score of 0–18, and a high Cronbach’s alpha coefficient (alpha = 0.843). This BRPS fills an important gap in the field of rabbit medicine and has the potential to improve the assessment and management of pain in rabbits providing veterinary professionals with a novel multidimensional pain assessment tool. Further studies will investigate the clinical utility, validity and reliability of the BRPS.

There is a need to ensure that the care and welfare for fish maintained in the laboratory are to the highest standards. This extends to the use of anaesthetics for both scientific study, humane killing and euthanasia at end of life. An anaesthetic should not induce negative behaviours and fish should not seek to avoid the anaesthetic. Surprisingly little information is available to facilitate a humane choice of anaesthetic agent for fish despite over 100 years of use and the millions of fish currently held in thousands of laboratories worldwide. Using a chemotaxic choice chamber we found different species specific behavioural responses among four closely related fish species commonly held in the laboratory, exposed to three widely used anaesthetic agents. As previously found for zebrafish ( Danio rerio ), the use of MS-222 and benzocaine also appears to induce avoidance behaviours in medaka ( Oryzias latipes ); but etomidate could provide an alternative choice. Carp ( Cyprinus carpio ), although closely related to zebrafish showed avoidance behaviours to etomidate, but not benzocaine or MS-222; and rainbow trout ( Oncorhynchus mykiss ) showed no avoidance to the three agents tested. We were unable to ascertain avoidance responses in fathead minnows ( Pimephales promelas ) and suggest different test paradigms are required for that species.

This study addresses a fundamental question in fish welfare: are the anaesthetics used for fish aversive? Despite years of routine general use of many agents, within both scientific research and aquaculture, there is a paucity of information regarding their tolerance and associated behavioural responses by fish. This study examined nine of the most commonly used fish anaesthetic agents, and performed preference tests using adult mixed sex zebrafish (Danio rerio), the most commonly held laboratory fish. Video tracking software quantified swimming behaviour related to aversion for each anaesthetic at 50% of its standard recommended dose compared with clean water in a flow-through chemotaxic choice chamber. Results suggest that several commonly used anaesthetics were aversive, including two of the most commonly recommended and used: MS222 (ethyl 3-aminobenzoate methanesulphate) and benzocaine. For ethical best practice, it is recommended that compounds that are aversive, even at low concentration, should no longer be used routinely for anaesthesia or indeed the first step of humane euthanasia of adult zebrafish. Two agents were found not to induce aversive behavioural responses: etomidate and 2,2,2 tribromoethanol. For the millions of adult zebrafish used in laboratories and breeding worldwide, etomidate appears best suited for future routine humane use.

Broiler (meat) chickens have been subjected to intense genetic selection. In the past 50 years, broiler growth rates have increased by over 300% (from 25 g per day to 100 g per day). There is growing societal concern that many broiler chickens have impaired locomotion or are even unable to walk. Here we present the results of a comprehensive survey of commercial flocks which quantifies the risk factors for poor locomotion in broiler chickens. We assessed the walking ability of 51,000 birds, representing 4.8 million birds within 176 flocks. We also obtained information on approximately 150 different management factors associated with each flock. At a mean age of 40 days, over 27.6% of birds in our study showed poor locomotion and 3.3% were almost unable to walk. The high prevalence of poor locomotion occurred despite culling policies designed to remove severely lame birds from flocks. We show that the primary risk factors associated with impaired locomotion and poor leg health are those specifically associated with rate of growth. Factors significantly associated with high gait score included the age of the bird (older birds), visit (second visit to same flock), bird genotype, not feeding whole wheat, a shorter dark period during the day, higher stocking density at the time of assessment, no use of antibiotic, and the use of intact feed pellets. The welfare implications are profound. Worldwide approximately 2 x 10(10) broilers are reared within similar husbandry systems. We identify a range of management factors that could be altered to reduce leg health problems, but implementation of these changes would be likely to reduce growth rate and production. A debate on the sustainability of current practice in the production of this important food source is required.

[This corrects the article DOI: 10.1371/journal.pone.0252417.].

Objective: Determine the accuracy of a novel technique for confirmation of the day of ovulation and prediction of ovulation in subsequent cycles for the purpose of conception using a skin-worn sensor in a population with ovulatory dysfunction. Methods: A total of 80 participants recorded consecutive overnight temperatures using a skin-worn sensor at the same time as a commercially available vaginal sensor for a total of 205 reproductive cycles. The vaginal sensor and its associated algorithm were used to determine the day of ovulation, and the ovulation results obtained using the skin-worn sensor and its associated algorithm were assessed for comparative accuracy alongside a number of other statistical techniques, with a further assessment of the same skin-derived data by means of the “three over six” rule. A number of parameters were used to divide the data into separate comparative groups, and further secondary statistical analyses were performed. Results: The skin-worn sensor and its associated algorithm (together labeled “SWS”) were 66% accurate for determining the day of ovulation (±1 day) or the absence of ovulation and 90% accurate for determining the fertile window (ovulation day ±3 days) in the total study population in comparison to the results obtained from the vaginal sensor and its associated algorithm (together labeled “VS”). Conclusion: SWS is a useful tool for confirming the fertile window and absence of ovulation (anovulation) in a population with ovulatory dysfunction, both known and determined by means of the timing of ovulation. The body site where the skin-worn sensor was worn (arm or wrist) did not appear to affect the accuracy. Prior diagnosis of known causes of ovulatory dysfunction appeared to affect the accuracy to a lesser extent than those cycles grouped into late ovulation and “early and normal ovulation” groups. SWS is a potentially useful tool for predicting ovulation in subsequent cycles, with greater accuracy obtained for the “normal ovulation” group.

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as ‘emotional fever’. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2–4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes.

Sessile animals, like corals, frequently suffer physical injury from a variety of sources, thus wound-healing mechanisms that restore tissue integrity and prevent infection are vitally important for defence. Despite the ecological importance of reef-building corals, little is known about the cells and processes involved in wound healing in this group or in phylogenetically basal metazoans in general. A histological investigation into wound healing of the scleractinian coral Porites cylindrica at 0 h, 6 h, 24 h and 48 h after injury revealed differences in cellular components between injured and healthy tissues. Cell counts of the obligate endosymbiont, Symbiodinium, and melanin volume fraction analysis revealed rapid declines in both Symbiodinium abundance and tissue cross-sectional area occupied by melanin-containing granular cells after injury. Four phases of wound healing were identified, which are similar to phases described for both vertebrates and invertebrates. The four phases included (i) plug formation via the degranulation of melanin-containing granular cells; (ii) immune cell infiltration (inflammation); (iii) granular tissue formation (proliferation); and (iv) maturation. This study provides detailed documentation of the processes involved in scleractinian wound healing for the first time and further elucidates the roles of previously-described immune cells, such as fibroblasts. These results demonstrate the conservation of wound healing processes from anthozoans to humans.

Venom producing animals are ubiquitously disseminated among vertebrates and invertebrates such as fish, snakes, scorpions, spiders, and ticks. Of the ~890 tick species worldwide, 27 have been confirmed to cause paralysis in mammalian hosts. The Australian paralysis tick ( Ixodes holocyclus ) is the most potent paralyzing tick species known. It is an indigenous three host tick species that secretes potent neurotoxins known as holocyclotoxins (HTs). Holocyclotoxins cause a severe and harmful toxicosis leading to a rapid flaccid paralysis which can result in death of susceptible hosts such as dogs. Antivenins are generally polyclonal antibody treatments developed in sheep, horses or camels to administer following bites from venomous creatures. Currently, the methods to prevent or treat tick paralysis relies upon chemical acaricide preventative treatments or prompt removal of all ticks attached to the host followed by the administration of a commercial tick-antiserum (TAS) respectively. However, these methods have several drawbacks such as poor efficacies, non-standardized dosages, adverse effects and are expensive to administer. Recently the I. holocyclus tick transcriptome from salivary glands and viscera reported a large family of 19 holocyclotoxins at 38-99% peptide sequence identities. A pilot trial demonstrated that correct folding of holocyclotoxins is needed to induce protection from paralysis. The immunogenicity of the holocyclotoxins were measured using commercial tick antiserum selecting HT2, HT4, HT8 and HT11 for inclusion into the novel cocktail vaccine. A further 4 HTs (HT1, HT12, HT14 and HT17) were added to the cocktail vaccine to ensure that the sequence variation among the HT protein family was encompassed in the formulation. A second trial comparing the cocktail of 8 HTs to a placebo group demonstrated complete protection from tick challenge. Here we report the first successful anti-venom vaccine protecting dogs from tick paralysis.